Pre-vaccination prevalence of high-risk human papillomaviruses (HPV) in women from Kosovo and their related sociodemographic characteristics

(1)

ORIGINAL PAPER / G y N E cO LO G y ISSN 0017–0011 DOI: 10.5603/GP.a2018.0083

Corresponding author:

Pranvera Zejnullahu Raçi

Department of Obstetrics and Gynecology, University Clinical Center of Kosovo, University “Hasan Prishtina”, Lagja e Spitalit n.n, 10000, Prishtina, Kosovo tel.: 37744111404

e-mail: pranveraz@yahoo.com

Pre-vaccination prevalence of high-risk human papillomaviruses (HPV) in women from Kosovo and

their related sociodemographic characteristics

Pranvera Zejnullahu Raçi

¹

, Lea Hošnjak

²

, Mario Poljak

²

, Snježana Židovec Lepej

³

, Adriana Vince

^4,5

1Department of Obstetrics and Gynecology, University Clinical Center of Kosovo, University “Hasan Prishtina”, Prishtina, Kosovo

2Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia

3Department of Molecular Diagnostics and Flow Cytometry, Dr. Fran Mihaljević University Hospital for Infectious Diseases Zagreb, Croatia

4Department of Viral Hepatitis, Dr. Fran Mihaljević University Hospital for Infectious Diseases, Zagreb, Croatia

5School of Medicine, University of Zagreb, Zagreb, Croatia

ABSTRACT

Objectives: Kosovo’s current health care system does not support organized nationwide cervical cancer screening and human papillomavirus (HPV) vaccination programs. To date, no reliable data are available on cervical cancer incidence and mortality in Kosovo, or on high-risk HPV (HR-HPV) prevalence and HPV type distribution. Our aim is to determinate the pre-vaccination prevalence and distribution of HR-HPVs and to assesses the associations between sociodemographic characteristics and increased risk of HPV infection in women from Kosovo.

Material and methods: Detection of HR-HPV DNA in cytologically evaluated cervical smears was performed using a clini- cally validated Abbott RealTime High Risk HPV test, Roche Linear Array HPV Genotyping Test, HPV52 type-specific real-time PCR and an in-house GP5+/GP6+/68 PCR.

Results: The crude overall prevalence of any of the HR-HPVs was estimated at 13.1% (26/199; 95% confidence interval (CI): 9.1–18.5%), with HPV16 being the most common type (7/26, 26.9%), followed by HPV31 and HPV51, each detected in 4/26 (15.4%) cervical specimens, HPV18, detected in 3/26 (11.5%) specimens, HPV52 and HPV66, each detected in 2/26 (7.7%) specimens, and HPV33, HPV45, HPV56, and HPV58, each detected in a single (3.9%) specimen. Women over 40 (OR = 0.36), older than 18 at sexual debut (odds ratio (OR) = 0.28), those that had delivered at least one child (OR = 0.32), and those that had a history of pregnancy termination (OR = 0.39) were at lower risk for HPV infection.

Conclusion: Because more than 70% of cervical precancerous lesions could have been prevented in Kosovo using nation- wide HPV-based cervical cancer screening and HPV vaccination, it is of outmost importance to implement both programs in the national health care system as soon as possible.

Key words: cervical cancer, high-risk human papillomaviruses, Kosovo, prevalence, vaccination

Ginekologia Polska 2018; 89, 9: 485–494

INTRODUCTION

Cervical cancer is the fourth most frequent cancer in women; in 2012 and 2015 there were an estimated 530,000 and 526,000 new cases worldwide, respectively, and it was responsible for 266,000 (7.5% of all cancer deaths)

and 239,000 (6.5% of all cancer deaths) deaths [1, 2]. In 2015, one in 68 (1.5%) women developed cervical cancer before age 79, with the odds being higher in low-income countries, where one in 24 (4.2%) women developed cervical cancer, in comparison to high-income countries, where one in

(2)

115 (0.9%) women developed cervical cancer during their lifetime [2]. Between 2005 and 2015, age-standardized incidence rates of cervical cancer decreased globally; however, the decline was less prominent in low-income countries [2].

Persistent infection with certain types of human papillomaviruses (HPV) is a necessary, but non-sufficient, etiological factor of cervical cancer [3, 4]. To date, 206 HPV types have been identified and officially recognized by the Interna- tional Human Papillomavirus Reference Center, located at the Karolinska Institute in Stockholm, Sweden (http://www.

hpvcenter.se/html/refclones.html). According to their clinical relevance, approximately 40 HPV types from the genus Alphapapillomavirus (Alpha-PV) are roughly subdivided into high-risk (HR) and low-risk (LR) HPV types. HR-HPVs (Group 1 carcinogens: HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, and HPV59) are etiologically associated with more than 98% of cervical cancers, 70 to 90% of anal and vaginal cancers, 40% of vulvar cancers, 47% of penile cancers, and 25 to 30% of oropharyngeal cancers. In addition, HPV68 is considered probably carcinogenic to humans (a Group 2A carcinogen), and HPV26, HPV30, HPV34, HPV53, HPV66, HPV67, HPV69, HPV70, HPV73, HPV82, HPV85, and HPV97 are classified as possibly carcinogenic to humans (Group 2B carcinogens). Low-risk HPV types (mainly HPV6 and HPV11) are associated with more than 90% of anogenital warts and laryngeal papillomas [3, 4].

Worldwide, the age-adjusted prevalence of HPV infection in women with normal cervical cytology is estimated at 10.4%, ranging from 8.1% in Europe to 22.1% in Africa [5].

Several global meta-analyses have suggested that the most prevalent HPV types in normal cervical cytological specimens are HPV16, HPV18, HPV31, HPV52, and HPV58 [5–7]. Moreo- ver, whereas HPV16 and HPV18 are etiologically associated with 71% of cervical cancer cases, this percentage rises to more than 90% when including infections with HPV6, HPV11, HPV31, HPV33, HPV45, HPV52, and HPV58 [4, 8, 9]. Because HPV prevalence and HPV type distribution vary greatly across different populations and geographical regions, regional HPV prevalence and type distribution studies are important before implementing HPV-based cervical cancer screening and HPV vaccination in particular countries or regions [5, 8].

Kosovo is a small low-income country with a population of 1.7 million in southeastern Europe that has recently experienced war and postwar difficulties. Kosovo’s current health care system does not support organized nationwide cervical cancer screening and HPV vaccination programs. In contrast to other countries in the region (e.g. Albania, Bul- garia, Bosnia and Herzegovina, Croatia, Macedonia, Monte- negro, Romania, and Serbia), no reliable data are available on cervical cancer incidence and mortality in Kosovo, or on HR-HPV prevalence and HPV type distribution. Thus, the objectives of this study were to determine for the first

time the pre-vaccination prevalence and distribution of HR-HPVs and to assess the associations between particular sociodemographic characteristics and increased risk of HPV infection in women from Kosovo.

MATERIAL AND METHODS Specimen and data collection

A total of 199 women, 18 to 63 years old, attending the outpatient unit of the Department of Gynecology and Ob- stetrics, from August 2015 to February 2016, were enrolled and had a valid sample for HPV testing. The cohort repre- sents female population of Kosovo attending the screening for cervical cancer. Namely outpatient clinic of the Univer- sity Clinical Centre of Kosovo (UCCK), based in Prishtina, where our study was performed is the only public institu- tion in Kosovo where the screening for cervical cancer and PAP-smear testing are offered to patients in whole country.

Patients included in the study were randomly selected from all patients visiting the outpatient Clinic, were the main purpose of the visit of the women was screening for cervical cancer or just routine gynaecological examination. All of the women provided written informed consent and completed a questionnaire on sociodemographic characteristics. Dur- ing a gynecological examination, one cervical smear was obtained for cytological examination, and another cervical smear was stored in 1 ml of Digene Specimen Transport Medium (STM) (Qiagen, Hilden, Germany) at −20 °C for subsequent HPV typing.

Detection of HPV DNA and HPV typing Detection of HR-HPV DNA was performed using a clinical- ly validated RealTime High Risk HPV test (RealTime) (Abbott, Wiesbaden, Germany), which enables concurrent individual typing for HPV16 and HPV18, as well as pooled detection of 12 other carcinogenic HPV types (HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, and HPV68), and also includes an internal process control for sample adequacy, DNA extraction, and amplification [10, 11].

In addition to several other transport media [10], the assay has also been clinically validated for use with cervical specimens collected with Digene STM (Qiagen) [12]. In specimens positive for other carcinogenic HPVs, HPV typing was subse- quently performed using the Linear Array HPV Genotyping Test (Roche LA) (Roche Molecular Diagnostics, Branchburg, NJ) and, if necessary, also with an HPV52 type-specific real-time PCR assay and an in-house GP5+/GP6+/68 PCR assay, as described previously [11, 13, 14].

Statistical analyses

Univariate and multiple logistic regression analyses were used to assess the associations between sociodemographic characteristics and overall HPV infection. A likelihood ratio

(3)

test was used to investigate the associations between sociodemographic characteristics and HR-HPV types. Associa- tions with p < 0.05 were treated as statistically significant.

Analyses were performed with SPSS, version 23.

Ethical statement

This study was performed in compliance with the Dec- laration of Helsinki and was approved by the Ethics and Professional Board of the Ministry of Health of the Republic of Kosovo (consent number 05-2826). All women included in the study provided written informed consents and only their sociodemographic characteristics were available to all researchers, preserving patients’ anonymity.

RESULTS

A total of 199 women, 20 to 63 years old, with a mean age of 41.8 years (SD ± 9.3 years) were included in the study (Tab.

1). The majority of women had graduated from a secondary school (107/199, 53.8%), were working as housewives or farmers (143/199, 71.9%) and were married (176/199, 88.4%), were a part of a small family (only parents and their children: 141/199, 70.9%) with a sufficient income (104/199, 52.3%), were at least 18 years old at the time of first sexual intercourse (168/199, 84.4%), had given birth to at least one child (178/199, 89.4%), had had at least one pregnancy terminated (100/199, 50.3%), had a regular menstrual cycle (126/199, 63.3%), were not using contraception (166/199, 83.4%), and were non-smokers (149/199, 74.9%) (Tab. 1).

The vast majority of women (165/199, 82.9%) had a normal cytological result (negative for intra-epithelial lesion or malignancy, NILM). Atypical squamous cells of undetermined significance (ASCUS) were detected in cervical specimens of 30/199 (15.1%) women and atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion (ASC-H), and cervical cancer were detected in two women, respectively (Tab. 1).

Detection and typing of HPV infection The crude overall prevalence of HR-HPV infection with any of the targeted 14 HPV types was estimated at 13.1%

(26/199, 95% confidence interval (95% CI): 9.1–18.5%) (Tab.

2). As shown in Table 3, infections with HPV16 were overall the most common, followed by HPV31 and HPV51, and HPV18. Similarly, HPV16 was also the most frequently detected HPV type in patients with NILM, followed by HPV31 and HPV51 (Tab. 3). Patients with ASCUS were most frequently infected with HPV18 (Tab. 3).

Both cervical cancer cases determined by the cytological examination were additionally confirmed using histopa- thology. Even though no multiple HR-HPV infections were determined in our study population, using Roche LA and an in-house GP5+/GP6+/68 PCR assay, single HPV16 and

HPV66-positive cervical specimens were additionally positive for HPV84 and HPV67, respectively, two HPV51-positive cervical specimens were additionally positive for HPV53 and HPV62, respectively, and the HPV58-positive cervical specimen was additionally positive for HPV6 and HPV73.

Association between sociodemographic characteristics and HPV infection

As shown in Table 4, using univariate logistic regression analysis to investigate potential differences in the sociodemographic characteristics of HPV-negative vs. HPV-positive patients, statistically significant associations with HPV infection were found only for women’s age (p = 0.015), age at the time of first sexual intercourse (p = 0.006), delivery (p = 0.026), and history of pregnancy termination (p = 0.033). Despite the fact that the preva- lence of HPV infection was the highest in women 30 to 39 years old (21.2% (11/52); mean: 20.8%; standard deviation SD: ± 6.4), followed by women 20 to 29 years old (19.1% (4/21); mean: 19.0%; SD: ± 10.5), 40 to 49 years old (9.6% (8/83); mean: 9.6%; SD: ± 4.2), and over 50 (7.0%

(3/43); mean: 7.0%; SD: ± 5.8) (Fig. 1), the differences were not statistically significant (p = 0.127). However, women over 40 had lower odds for HPV infection (odds ratio (OR) = 0.36; 95% CI: 0.15–0.84) (Tab. 4). Moreover, lower odds for HPV infection were also observed among women that were at least 18 years old at sexual debut (OR = 0.28;

95% CI: 0.11–0.69), had given birth to at least one child (OR = 0.32; 95% CI: 0.11–0.91), or had a history of pregnancy termination (OR = 0.39; 95% CI: 0.16–0.95) (Tab. 4).

Due to the small number of units in the category of the de- pendent variable (HPV infection), only three independent variables (age at inclusion in the study: up to 40 years old and over 40, age at first sexual intercourse: below 18 and at least 18 years old or older, and history of delivering at least one child), were included in the multiple logistic

Age [years]

HPV prevalence [%]

35.0 30.0 25.0 20.0 15.0 10.0 5.0

0.0 20−29 30−39 40−49 50+

Figure 1. Age-specific HPV prevalence with standard deviation among 199 women from Kosovo included in the study

(4)

Table 1. Socio-demographic characteristics of women included in the study and the results of cytology

N = 199 (%)

(Mean) age (SD) 41.8 (9.3)

< 40 years old 79 (39.7)

> 40 years old 120 (60.3)

Education

Finished elementary school or less 51 (25.6)

Finished secondary school 107 (53.8)

More than secondary school 41 (20.6)

Employment status

Employed 51 (25.6)

Housewife or farmer 143 (71.9)

Student 5 (2.5)

Marital status

Married 176 (88.4)

Divorced/widow 13 (6.5)

Single 10 (5.0)

Family size

Single 4 (2.0)

Small family (parents&children) 141 (70.9)

Large family (parents, children and other family members) 54 (27.1)

Family income*

Insufficient 54 (27.1)

Sufficient 104 (52.3)

Good 41 (20.6)

Age at first sexual intercourse

< 18 years old 31 (15.6)

≥ 18 years old 168 (84.4)

Delivery

Yes 178 (89.4)

No 21 (10.6)

Pregnancy termination

Yes 100 (50.3)

No 99 (49.7)

Menstrual cycle

Regular 126 (63.3)

Irregular 40 (20.1)

Postmenopausal 33 (16.6)

Contraception use

Yes 33 (16.5)

No 166 (83.4)

Smoking

Yes 50 (25.1)

No 149 (74.9)

Cytology

Negative for intra-epithelial lesion or malignancy (NILM) 165 (82.9)

Atypical squamous cells of undetermined significance (ASCUS) 30 (15.1)

Atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion (ASC-H) 2 (1.0)

Cervical cancer 2 (1.0)

Categorical and continuous variables are shown as frequencies (percentages) and as mean (standard deviation), respectively. * The family’s financial situation was evaluated by patients themselves.

(5)

Table 2. Overall prevalence of HR-HPVs among 199 women from Kosovo according to the cytology results

HPV types All women (N = 199) Cytology results

N Prevalence (%)

(95% CI) NILM (N = 165) ASCUS (N = 30) ASC-H (N = 2) Cervical cancer (N = 2) N Prevalence (%)

(95% CI) N Prevalence (%)

(95% CI) Any HR-HPV 26 13.1 (9.1–18.5) 16 9.7 (6.1–15.2) 7 23.3 (11.8–40.9) 1 50.0 (9.5–90.6) 2 100 (34.2–100) HPV16/HPV18 10 5.0 (2.8–9.0) 5 3.0 (1.3–6.9) 2 6.7 (1.9–21.3) 1 50.0 (9.5–90.6) 2 100 (34.2–100)

HPV16 7 3.5 (1.7–7.1) 5 3.0 (1.3–6.9) 0 0 (–) 1 50.0 (9.5–90.6) 1 50.0 (9.5–90.5)

HPV18 3 1.5 (0.5–4.3) 0 0 (–) 2 6.7 (1.9–21.3) 0 0 (–) 1 50.0 (9.5–90.5)

HPV31 4 2.0 (0.8–5.1) 3 1.8 (0.6–5.2) 1 3.3 (0.6–16.7) 0 0 (–) 0 0 (–)

HPV33 1 0.5 (0.1–2.8) 1 0.6 (0.1–3.4) 0 0 (–) 0 0 (–) 0 0 (–)

HPV45 1 0.5 (0.1–2.8) 1 0.6 (0.1–3.4) 0 0 (–) 0 0 (–) 0 0 (–)

HPV51 4 2.0 (0.8–5.1) 3 1.8 (0.6–5.2) 1 3.3 (0.6–16.7) 0 0 (–) 0 0 (–)

HPV52 2 1.0 (0.3–3.6) 1 0.6 (0.1–3.4) 1 3.3 (0.6–16.7) 0 0 (–) 0 0 (–)

HPV56 1 0.5 (0.1–2.8) 1 0.6 (0.1–3.4) 0 0 (–) 0 0 (–) 0 0 (–)

HPV58 1 0.5 (0.1–2.8) 0 0 (–) 1 3.3 (0.6–16.7) 0 0 (–) 0 0 (–)

HPV66 2 1.0 (0.3–3.6) 1 0.6 (0.1–3.4) 1 3.3 (0.6–16.7) 0 0 (–) 0 0 (–)

Abbreviations — NILM (negative for intra-epithelial lesion or malignancy); ASCUS — atypical squamous cells of undetermined significance; ASC-H — atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion; 95% CI — 95% confidence interval

Table 3. Distribution of HR-HPV types among 26 women infected with any of the 14 HR-HPVs according to the cytology results

HPV types N % Cytology results

NILM ASCUS ASC-H Cervical cancer

N % N % N % N %

Any HR-HPV 26 100 16 100 7 100 1 100 2 100

HPV16/HPV18 10 38.5 5 31.3 2 28.6 1 100 2 100

HPV16 7 26.9 5 31.3 0 0 1 100 1 50.0

HPV18 3 11.5 0 0 2 28.6 0 0 1 50.0

HPV31 4 15.4 3 18.8 1 14.3 0 0 0 0

HPV33 1 3.9 1 6.3 0 0 0 0 0 0

HPV45 1 3.9 1 6.3 0 0 0 0 0 0

HPV51 4 15.9 3 18.8 1 14.3 0 0 0 0

HPV52 2 7.7 1 6.3 1 14.3 0 0 0 0

HPV56 1 3.9 1 6.3 0 0 0 0 0 0

HPV58 1 3.9 0 0 1 14.3 0 0 0 0

HPV66 2 7.7 1 6.3 1 14.3 0 0 0 0

Abbreviations — NILM (negative for intra-epithelial lesion or malignancy); ASCUS — atypical squamous cells of undetermined significance); ASC-H — atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion

regression analysis. When controlling for other varia bles in the model, all three independent variables were statistically significantly associated with HPV infection, with OR = 0.37 (95% CI: 0.15–0.94) and p = 0.036 for overall older age (over 40 years old), OR = 0.18 (95% CI: 0.06–0.50) and p < 0.001 for older age at sexual debut (at least 18 years

old), and OR = 0.28 (95% CI: 0.09–0.92) and p = 0.035 for history of delivering at least one child. The results of the multiple logistic regression analysis therefore suggest that women younger than 40, who were younger than 18 at the time of first sexual intercourse and that have not yet given birth, are at greater risk for HPV infection [Fig. 1].

(6)

Table 4. Association between socio-demographic characteristics and HPV infection (results of an univariate logistic regression analysis) HPV-negative (n = 173) HPV-positive (n = 26) OR (95% CI) P-value

(Mean) age (SD) 42.4 (9.2) 38.4 (9.9) 0.96 (0.91; 1) 0.044

< 40 years old 63 (36.4) 16 (61.5) 1

> 40 years old 110 (63.6) 10 (38.5) 0.36 (0.15; 0.84) 0.015

Education

Finished elementary school or less 45 (26) 6 (23.1) 1

Finished secondary school 92 (53.2) 15 (57.7) 1.22 (0.44; 3.36) 0.697

More than secondary school 36 (20.8) 5 (19.2) 1.04 (0.29; 3.69) 0.950

Employed 44 (25.4) 7 (26.9) 1

Housewife or farmer 125 (72.3) 18 (69.2) 0.91 (0.35; 2.31) 0.835

Student 4 (2.3) 1 (3.8) 1.57 (0.15; 6.18) 0.702

Marital status

Divorced/widowed 10 (5.8) 3 (11.5) 1

Married 155 (89.6) 21 (80.8) 0.45 (0.11; 1.77) 0.244

Single 8 (4.6) 2 (7.7) 0.83 (0.11; 6.26) 0.859

Family size

Single 3 (1.7) 1 (3.8) 1

Small family (parents&children) 124 (71.7) 17 (65.4) 0.41 (0.04; 4.18) 0.453

Large family (parents, children and other

family members) 46 (26.6) 8 (30.8) 0.52 (0.05; 5.66) 0.593

Family income*

Insufficient 47 (27.2) 7 (26.9) 1

Sufficient 88 (50.9) 16 (61.5) 1.22 (0.47; 3.18) 0.683

Good 38 (22) 3 (11.5) 0.53 (0.13; 2.19) 0.380

(Mean) age (SD) at first sexual intercourse 21.9 (4.2) 20.8 (5.4) 0.94 (0.84; 1.04) 0.230

< 18 years old 22 (12.7) 9 (34.6) 1

> 18 years old 151 (87.3) 17 (65.4) 0.28 (0.11; 0.69) 0.006

Delivery

No 15 (8.7) 6 (23.1) 1

Yes 158 (91.3) 20 (76.9) 0.32 (0.11; 0.91) 0.026

No 81 (46.8) 18 (69.2) 1

Yes 92 (53.2) 8 (30.8) 0.39 (0.16; 0.95) 0.033

Menstrual cycle

Irregular 33 (19.1) 7 (26.9) 1

Regular 108 (62.4) 18 (69.2) 0.79 (0.30; 2.04) 0.620

Postmenopausal 32 (18.5) 1 (3.8) 0.15 (0.02; 1.27) 0.081

Contraception

No 145 (83.8) 21 (80.8) 1

Yes 28 (16.2) 5 (19.2) 1.23 (0.43; 3.54) 0.697

Smoking

No 131 (75.7) 18 (69.2) 1

Yes 42 (24.3) 8 (30.8) 1.39 (0.56; 3.42) 0.477

Categorical and continuous variables are shown as frequencies (percentages) and as mean (standard deviation), respectively. * The family’s financial situation was evaluated by patients themselves. Abbreviations — OR (odds ratio); 95% CI — 95% confidence interval

(7)

Association between sociodemographic characteristics and HR-HPV types

As shown in Table 5, the results of the likelihood ratio test suggested that women infected with HPV16 were older in comparison to women infected with other HR-HPV types (p = 0.037). In addition, all women infected with HPV16 have given birth to at least one child, whereas only 68.4% of women infected with other HR-HPVs had biological chil- dren (p = 0.036). Other characteristics were not statistically significantly associated with specific HR-HPV types.

DISCUSSION

Infections with over 40 HPV types from the clinically most important Alpha-PV genus are considered to be the most frequently sexually transmitted infections in both genders. In addition, worldwide, up to 4.5% of incident cancer cases, including cervical, anogenital, and head and neck cancers, are etiologically associated with HPV infection.

Moreover, cervical cancer accounts for 83% of all HPV-related cancers, with the majority occurring in women originat- ing from less-developed countries [4].

In the female population from Kosovo, the crude overall prevalence of infection with any of the 14 HR-HPVs was estimated at 13.1%. Interestingly, in our study population the overall HR-HPV prevalence in NILM was lower than in studies on a total of 10.744 eligible women from other countries in central and eastern Europe (9.7%; 95% CI: 6.1–15.2%

vs. 18.0%; 95% CI: 17.0–19.0%, respectively) [15–23], with reported high incidence rates of cervical cancer and related mortality rates [1].Our results were concordant with those obtained in the largest study in the region to date, performed on 4.199 Slovenian women (HR-HPV prevalence of 10.7%; cervical cancer incidence rate per 100,000: 13.4;

mortality rate due to cervical cancer per 100,000: 6.1) [1, 14], as well as with global data (HR-HPV prevalence of 10.4%;

cervical cancer incidence rate per 100,000: 15.1; mortality rate due to cervical cancer per 100,000: 7.6) [1, 5].

Three prophylactic HPV vaccines are commercially available: a bivalent HPV16/18 vaccine (Cervarix;

GlaxoSmithKline Biologicals, Belgium), a quadrivalent HPV6/11/16/18 vaccine (Gardasil/Silgard; Merck & Co., USA / Sanofi Pasteur MSD, France), and a nonavalent HPV6/11/16/18/31/33/45/52/58 vaccine (Gardasil9, Merck

& Co.).After the first 10 years of routine use of HPV vaccines, comprehensive clinical trials and real-life data have confirmed the vaccines’ safety and effectiveness.

Even though the introduction of HPV vaccination has the potential to substantially reduce the incidence of cervical cancer cases, the full effect on women of all ages will be de- tectable no sooner than after 30 years. Thus well-organized cervical cancer screening programs will still play a crucial role in the prevention of cervical cancer and bridging the

gap until the full effect of HPV vaccines can be observed [24]. Vaccarella et al. estimated recently that, with the implementation of well-organized cervical cancer screening by 2017 in six Baltic, central European, and eastern Euro- pean countries (Estonia, Lithuania, Latvia, Belarus, Bulgaria, and Russia), in which cervical cancer screening is absent or, at best, opportunistic with low coverage and quality, 180,000 new cervical cancer cases could be prevented from 2017 to 2040 [24]. In comparison to cytology-based screening, HPV-based screening is more effective and efficient for the prevention of invasive cervical cancer. Therefore, HPV testing is an invaluable part of guidelines for cervical cancer screening, triage, and follow-up after treatment [24].

Even though more than 190 distinct HPV tests and at least 127 test variants were commercially available in 2015, only a fraction of HPV tests fulfill the criteria for use in primary cervical cancer screening, with RealTime, used in this study, being one of them [25]. The World Health Organization sug- gests that, in countries without established cytology-based cervical cancer screening, HPV-based screening programs, in combination with the implementation of HPV vaccination, could further accelerate screening benefits and reduce the burden of cervical cancer [24].

Despite the fact that a comprehensive strategy, including HPV vaccination and HPV-based cervical cancer screening, has been demonstrated to be cost-effective in nearly all countries [26], progress toward prevention is often hindered due to relatively low access to vaccines [27] and limited use of cervical cancer screening [28], especially in low-income countries.

We hope that the results of this study will also persuade the authorities to implement nationwide cervical cancer screening and HPV vaccination programs in Kosovo.

Even though multiple HPV infections are not uncom- mon in cervical specimens, each HPV type is associated with a biologically separate and independent cervical lesion, as observed using laser capture micro-dissection (LCM) with HPV PCR typing (LCM-PCR) [29].In our study, multiple HPV types were detected in five cervical smears (HPV16+HPV84, HPV66+HPV67, HPV51+53, HPV51+62, and HPV6+58+73).

Although the cycle threshold values obtained using the RealTime were higher for HR-HPV types in comparison to other HPVs, suggesting the predominance of HR-HPVs, it is impossible to speculate on the etiological associations because the LCM-PCR technique cannot be used on smear samples.

Because persistent infection with HR-HPVs is a necessary, but non-sufficient, etiological factor of cervical cancer, several contributory factors have been described previously [3, 4, 30]. In this study, the results of the univariate and multiple logistic regression analyses suggested that women over 40 (univariate logistic regression: p = 0.015, OR = 0.36;

(8)

Table 5. Association between socio-demographic characteristics and HR-HPV types (results of likelihood ratio test) Other HPV type (n = 19) HPV16 (n = 7) P-value Age

< 40 years old 14 (73.7) 2 (28.6) 0.037

> 40 years old 5 (26.3) 5 (71.4)

Education

Finished elementary school or less 6 (31.6) 0 (0) 0.107

Finished secondary school 10 (52.6) 5 (71.4)

More than secondary school 3 (15.8) 2 (28.6)

Eemployed 5 (26.3) 2 (28.6) 0.725

Housewife or farmer 13 (68.4) 5 (71.4)

Student 1 (5.3) 0 (0)

Marital status

Married 15 (78.9) 6 (85.7) 0.303

Divorced/widowed 3 (15.8) 0 (0)

Single 1 (5.3) 1 (14.3)

Family size

Single 1 (5.3) 0 (0) 0.706

Small (parents&children) 12 (63.2) 5 (71.4)

Large (parents, children and other family

members) 6 (31.6) 2 (28.6)

Family income*

Insufficient 6 (31.6) 1 (14.3) 0.652

Sufficient 11 (57.9) 5 (71.4)

Good 2 (10.5) 1 (14.3)

Age at first sexual intercourse

< 18 7 (36.8) 2 (28.6) 0.691

> 18 12 (63.2) 5 (71.4)

Delivery

Yes 13 (68.4) 7 (100) 0.036

No 6 (31.6) 0 (0)

Yes 6 (31.6) 2 (28.6) 0.882

No 13 (68.4) 5 (71.4)

Menstrual cycle

Regular 13 (68.4) 5 (71.4) 0.725

Irregular 5 (26.3) 2 (28.6)

Postmenopausal 1 (5.3) 0 (0)

Contraception

Yes 4 (21.1) 1 (14.3) 0.691

No 15 (78.9) 6 (85.7)

Smoking

Yes 6 (31.6) 2 (28.6) 0.882

No 13 (68.4) 5 (71.4)

* The family’s financial situation was evaluated by patients themselves.

(9)

multiple logistic regression: p = 0.036, OR = 0.37), who were older than 18 at sexual debut (univariate logistic regression:

p = 0.006, OR = 0.28; multiple logistic regression: p < 0.001, OR = 0.18), and those with a history of delivering at least one child (univariate logistic regression: p = 0.026, OR = 0.32;

multiple logistic regression: p = 0.035, OR = 0.28) were at lower risk for HPV infection. The results of the univariate logistic regression analysis additionally suggested that women with a history of pregnancy termination also have lower odds (p = 0.033, OR = 0.39) for HPV infection. Our results are concordant with global data (reviewed in 30), reporting that the incidence/prevalence of HPV infection peaks at a younger age (below 25–35 years), soon after the start of sexual activity, and declines with age due to the spontane- ous clearance of HPV infections. Similarly as in our study, associations between cervical cancer, cervical intraepithelial lesions, and increasing number of pregnancies and parity were also found in previous case-control studies (reviewed in 30). Interestingly, according to the likelihood ratio test, in comparison to women infected with other HR-HPV types, women infected with HPV16 were older (p = 0.037) and have given birth to at least one child (p = 0.036).

CONCLUSIONS

In conclusion, this study is the first study reporting the HR-HPV prevalence and distribution of HPV types in Kosovo.

Because more than 70% of cervical precancerous lesions identified in the study could have been prevented using nationwide HPV-based cervical cancer screening and HPV vaccination programs, it is of outmost importance to implement both programs in the national health care system in Kosovo as soon as possible.

Acknowledgements

The authors would like to thank Suzana Manxhuka-Kerliu from the Institute of Pathology, University Clinical Center of Kosovo, University of Prishtina, Prishtina, for cytological examination of cervical specimens, Blanka Kušar from the Institute of Microbiology and Immunology, Faculty of Me- dicine, University of Ljubljana, for technical assistance, and Vanja Erčulj from RHO Sigma, Ljubljana, Slovenia, for help with statistical analyses.

Conflicts of interest

The authors have no conflicts of interest to declare.

REFERENCES

1. Ferlay J, Soerjomataram I, Dikshit R, et al. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012.

Int J Cancer. 2015; 136(5): E359–E386, doi: 10.1002/ijc.29210, indexed in Pubmed: 25220842.

2. Fitzmaurice C, Allen C, Barber RM, et al. Global Burden of Disease Cancer Collaboration. Global, Regional, and National Cancer Incidence, Mortal- ity, Years of Life Lost, Years Lived With Disability, and Disability-Adjusted

Life-years for 32 Cancer Groups, 1990 to 2015: A Systematic Analysis for the Global Burden of Disease Study. JAMA Oncol. 2017; 3(4): 524–548, doi: 10.1001/jamaoncol.2016.5688, indexed in Pubmed: 27918777.

3. Bouvard V, Baan R, Straif K, et al. WHO International Agency for Research on Cancer Monograph Working Group. A review of human carcinogens- -Part B: biological agents. Lancet Oncol. 2009; 10(4): 321–322, indexed in Pubmed: 19350698.

4. de Martel C, Plummer M, Vignat J, et al. Worldwide burden of cancer attributable to HPV by site, country and HPV type. Int J Cancer.

2017; 141(4): 664–670, doi: 10.1002/ijc.30716, indexed in Pubmed:

28369882.

5. de Sanjosé S, Diaz M, Castellsagué X, et al. Worldwide prevalence and genotype distribution of cervical human papillomavirus DNA in women with normal cytology: a meta-analysis. Lancet Infect Dis. 2007; 7(7):

453–459, doi: 10.1016/S1473-3099(07)70158-5, indexed in Pubmed:

17597569.

6. Bruni L, Diaz M, Castellsagué X, et al. Cervical human papillomavirus prevalence in 5 continents: meta-analysis of 1 million women with normal cytological findings. J Infect Dis. 2010; 202(12): 1789–1799, doi:

10.1086/657321, indexed in Pubmed: 21067372.

7. Guan P, Howell-Jones R, Li Ni, et al. Human papillomavirus types in 115,789 HPV-positive women: a meta-analysis from cervical infection to cancer. Int J Cancer. 2012; 131(10): 2349–2359, doi: 10.1002/ijc.27485, indexed in Pubmed: 22323075.

8. Li Ni, Franceschi S, Howell-Jones R, et al. Human papillomavirus type distribution in 30,848 invasive cervical cancers worldwide: Variation by geographical region, histological type and year of publication. Int J Cancer. 2011; 128(4): 927–935, doi: 10.1002/ijc.25396, indexed in Pubmed: 20473886.

9. Alemany L, de Sanjosé S, Tous S, et al. RIS HPV TT Study Group. Time trends of human papillomavirus types in invasive cervical cancer, from 1940 to 2007. Int J Cancer. 2014; 135(1): 88–95, doi: 10.1002/ijc.28636, indexed in Pubmed: 24382655.

10. Huang S, Tang N, Mak WB, et al. Principles and analytical performance of Abbott RealTime High Risk HPV test. Journal of Clinical Virology. 2009;

45: S13–S17, doi: 10.1016/s1386-6532(09)70003-4.

11. Poljak M, Ostrbenk A, Seme K, et al. Comparison of clinical and analytical performance of the Abbott Realtime High Risk HPV test to the performance of hybrid capture 2 in population-based cervical cancer screening. J Clin Microbiol. 2011; 49(5): 1721–1729, doi: 10.1128/JCM.00012-11, indexed in Pubmed: 21430098.

12. Carozzi FM, Burroni E, Bisanzi S, et al. Comparison of clinical performance of Abbott RealTime High Risk HPV test with that of hybrid capture 2 assay in a screening setting. J Clin Microbiol. 2011; 49(4): 1446–1451, doi:

10.1128/JCM.02311-10, indexed in Pubmed: 21325553.

13. Kocjan BJ, Poljak M, Seme K. Universal ProbeLibrary based real-time PCR assay for detection and confirmation of human papillomavirus genotype 52 infections. J Virol Methods. 2010; 163(2): 492–494, doi: 10.1016/j.

jviromet.2009.10.024, indexed in Pubmed: 19883693.

14. Učakar V, Poljak M, Klavs I. Pre-vaccination prevalence and distribution of high-risk human papillomavirus (HPV) types in Slove- nian women: a cervical cancer screening based study. Vaccine.

2012; 30(2): 116–120, doi: 10.1016/j.vaccine.2011.10.066, indexed in Pubmed: 22080175.

15. Poljak M, Seme K, Maver PJ, et al. Human papillomavirus prevalence and type-distribution, cervical cancer screening practices and current status of vaccination implementation in Central and Eastern Europe. Vaccine.

2013; 31 Suppl 7: H59–H70, doi: 10.1016/j.vaccine.2013.03.029, indexed in Pubmed: 24332298.

16. Kovachev S, Slavov V, Kovachev S, et al. Prevalence of human papillomavirus infection in women in some cities and regions of Bulgaria.

J Med Virol. 2013; 85(9): 1577–1584, doi: 10.1002/jmv.23652, indexed in Pubmed: 23852682.

17. Tachezy R, Smahelova J, Kaspirkova J, et al. Human papillomavirus type-specific prevalence in the cervical cancer screening population of Czech women. PLoS One. 2013; 8(11): e79156, doi: 10.1371/journal.

pone.0079156, indexed in Pubmed: 24265750.

18. Moga MA, Irimie M, Oanta A, et al. Type-specific prevalence of human papillomavirus by cervical cytology among women in Brasov, Romania. Asian Pac J Cancer Prev. 2014; 15(16): 6887–6892, indexed in Pubmed: 25169541.

19. Duvlis S, Popovska-Jankovic K, Arsova ZS, et al. HPV E6/E7 mRNA versus HPV DNA biomarker in cervical cancer screening of a group of Macedonian women. J Med Virol. 2015; 87(9): 1578–1586, doi:

10.1002/jmv.24199, indexed in Pubmed: 25880030.

(10)

20. Simanaviciene V, Gudleviciene Z, Popendikyte V, et al. Studies on the prevalence of oncogenic HPV types among Lithuanian women with cervical pathology. J Med Virol. 2015; 87(3): 461–471, doi:

10.1002/jmv.24073, indexed in Pubmed: 25196501.

21. Kovacevic G, Nikolic N, Jovanovic-Galovic A, et al. Frequency of twelve carcinogenic human papilloma virus types among women from the South Backa region, Vojvodina, Serbia. Turk J Med Sci. 2016; 46(1):

97–104, doi: 10.3906/sag-1410-47, indexed in Pubmed: 27511341.

22. Staykova J, Belovska T, Murad A, et al. Cervical Viral Infections among Asymptomatic Bulgarian Women. Cent Eur J Public Health. 2016; 24(3):

176–179, doi: 10.21101/cejph.a4299, indexed in Pubmed: 27760284.

23. Sabol I, Milutin Gašperov N, Matovina M, et al. Cervical HPV type-specific pre-vaccination prevalence and age distribution in Croatia. PLoS One.

2017; 12(7): e0180480, doi: 10.1371/journal.pone.0180480, indexed in Pubmed: 28692681.

24. Vaccarella S, Franceschi S, Zaridze D, et al. Preventable fractions of cervical cancer via effective screening in six Baltic, central, and eastern European countries 2017-40: a population-based study. Lancet Oncol.

2016; 17(10): 1445–1452, doi: 10.1016/S1470-2045(16)30275-3, indexed in Pubmed: 27567054.

25. Poljak M, Kocjan B, Oštrbenk A, et al. Commercially available molecular tests for human papillomaviruses (HPV): 2015 update. Journal of Clinical Virology. 2016; 76: S3–S13, doi: 10.1016/j.jcv.2015.10.023.

26. Jit M, Brisson M, Portnoy A, et al. Cost-effectiveness of female human papillomavirus vaccination in 179 countries: a PRIME modelling study. Lancet Glob Health. 2014; 2(7): e406–e414, doi:

10.1016/S2214-109X(14)70237-2, indexed in Pubmed: 25103394.

27. Bruni L, Diaz M, Barrionuevo-Rosas L, et al. Global estimates of human papillomavirus vaccination coverage by region and income level: a pooled analysis. Lancet Glob Health. 2016; 4(7):

e453–e463, doi: 10.1016/S2214-109X(16)30099-7, indexed in Pubmed: 27340003.

28. Santesso N, Mustafa RA, Schünemann HJ, et al. Guideline Support Group.

World Health Organization Guidelines for treatment of cervical intraepithelial neoplasia 2-3 and screen-and-treat strategies to prevent cervical cancer. Int J Gynaecol Obstet. 2016; 132(3): 252–258, doi: 10.1016/j.

ijgo.2015.07.038, indexed in Pubmed: 26868062.

29. Quint W, Jenkins D, Molijn A, et al. One virus, one lesion--individual components of CIN lesions contain a specific HPV type. J Pathol.

2012; 227(1): 62–71, doi: 10.1002/path.3970, indexed in Pubmed:

22127961.

30. Gargano JW, Nisenbaum R, Lee DR, et al. Age-group differences in human papillomavirus types and cofactors for cervical intraepithelial neoplasia 3 among women referred to colposcopy. Cancer Epidemiol Biomarkers Prev. 2012; 21(1): 111–121, doi: 10.1158/1055-9965.EPI-11- 0664, indexed in Pubmed: 22028398.